Abstract
Multicellular organisms achieve greater complexity through cell divisions that generate different cell types. We engineered a simple genetic circuit that induces asymmetric cell division and subsequent cell differentiation in Escherichia coli. The circuit involves a scaffolding protein, PopZ, that is stably maintained at a single cell pole over multiple asymmetric cell divisions. PopZ was functionalized to degrade the signaling molecule, c-di-GMP. By regulating synthesis of functionalized PopZ via small molecules or light, we can chemically or optogenetically control the relative abundance of two distinct cell types, characterized by either low or high c-di-GMP levels. Differences in c-di-GMP levels can be transformed into genetically programmable differences in protein complex assembly or gene expression, which in turn produce differential behavior or biosynthetic activities. This study shows emergence of complex biological phenomena from a simple genetic circuit and adds programmable bacterial cell differentiation to the genetic toolbox of synthetic biology and biotechnology.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.
References
Inaba, M. & Yamashita, Y. M. Asymmetric stem cell division: precision for robustness. Cell Stem Cell 11, 461–469 (2012).
Kysela, D. T., Brown, P. J., Huang, K. C. & Brun, Y. V. Biological consequences and advantages of asymmetric bacterial growth. Annu. Rev. Microbiol. 67, 417–435 (2013).
Ehrle, H. M. et al. Polar Organizing Protein PopZ Is Required for Chromosome Segregation in Agrobacterium tumefaciens. J. Bacteriol. 199, e00111–17 (2017).
Tan, I. S. & Ramamurthi, K. S. Spore formation in Bacillus subtilis. Environ. Microbiol. Rep. 6, 212–225 (2014).
Tsokos, C. G. & Laub, M. T. Polarity and cell fate asymmetry in Caulobacter crescentus. Curr. Opin. Microbiol. 15, 744–750 (2012).
Grünenfelder, B. et al. Proteomic analysis of the bacterial cell cycle. Proc. Natl Acad. Sci. USA 98, 4681–4686 (2001).
Abdelrahman, Y., Ouellette, S. P., Belland, R. J. & Cox, J. V. Polarized cell division of Chlamydia trachomatis. PLoS Pathog. 12, 1–20 (2016).
Schumacher, D. & Søgaard-Andersen, L. Regulation of motility and polarity in Myxococcus xanthus. Annu. Rev. Microbiol. 71, 61–78 (2017).
Logsdon, M. M. & Aldridge, B. B. Stable regulation of cell cycle events in mycobacteria: insights from inherently heterogeneous bacterial populations. Front. Microbiol. 9, 1–15 (2018).
Lyons, N. A. & Kolter, R. On the evolution of bacterial multicellularity. Curr. Opin. Microbiol. 24, 21–28 (2015).
Werner, J. N. et al. Quantitative genome-scale analysis of protein localization in an asymmetric bacterium. Proc. Natl Acad. Sci. USA 106, 7858–7863 (2009).
Eichenberger, P. et al. The program of gene transcription for a single differentiating cell type during sporulation in Bacillus subtilis. PLoS Biol. 2, 1664–1683 (2004).
Bowman, G. R. et al. Oligomerization and higher-order assembly contribute to sub-cellular localization of a bacterial scaffold. Mol. Microbiol. 90, 776–795 (2013).
Coquel, A. S. et al. Localization of protein aggregation in Escherichia coli is governed by diffusion and nucleoid macromolecular crowding effect. PLoS Comput. Biol. 9, 1–14 (2013).
Neeli-Venkata, R. et al. Robustness of the process of nucleoid exclusion of protein aggregates in Escherichia coli. J. Bacteriol. 198, 898–906 (2016).
Ebersbach, G., Briegel, A., Jensen, G. J. & Jacobs-Wagner, C. A self-associating protein critical for chromosome attachment, division, and polar organization in Caulobacter. Cell 134, 956–968 (2008).
Holmes, J. A. et al. Caulobacter PopZ forms an intrinsically disordered hub in organizing bacterial cell poles. Proc. Natl Acad. Sci. USA 113, 12490–12495 (2016).
Scheu, K., Gill, R., Saberi, S., Meyer, P. & Emberly, E. Localization of aggregating proteins in bacteria depends on the rate of addition. Front. Microbiol. 5, 1–5 (2014).
Simm, R., Morr, M., Kader, A., Nimtz, M. & Römling, U. GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessibility to motility. Mol. Microbiol 53, 1123–1134 (2004).
Ryu, M. & Gomelsky, M. Near-infrared light responsive synthetic c-di-GMP module for optogenetic applications. ACS Synth. Biol. 11, 802–810 (2013).
Jenal, U., Reinders, A. & Lori, C. Cyclic di-GMP: Second messenger extraordinaire. Nat. Rev. Microbiol. 15, 271–284 (2017).
Chou, S. H. & Galperin, M. Y. Diversity of cyclic di-GMP-binding proteins and mechanisms. J. Bacteriol. 198, 32–46 (2016).
Schmidl, S. R., Sheth, R. U., Wu, A. & Tabor, J. J. Refactoring and optimization of light-switchable Escherichia coli two-component systems. ACS Synth. Biol. 3, 820–831 (2014).
Tabor, J. J., Levskaya, A. & Voigt, C. A. Multichromatic control of gene expression in Escherichia coli. J. Mol. Biol. 405, 315–324 (2011).
Ryjenkov, Da, Simm, R., Römling, U. & Gomelsky, M. The PilZ domain is a receptor for the second messenger c-di-GMP: the PilZ domain protein YcgR controls motility in Enterobacteria. J. Biol. Chem. 281, 30310–30314 (2006).
Boehm, A. et al. Second messenger-mediated adjustment of bacterial swimming velocity. Cell 141, 107–116 (2010).
Chin, K. H. et al. Structural polymorphism of c-di-GMP bound to an EAL domain and in complex with a type II PilZ-domain protein. Acta Crystallogr. Sect. D. 68, 1380–1392 (2012).
Cabantous, S. et al. A new protein-protein interaction sensor based on tripartite split-GFP association. Sci. Rep. 3, 2854 (2013).
Shekhawat, S. S. & Ghosh, I. Split-protein systems: beyond binary protein-protein interactions. Curr. Opin. Chem. Biol. 15, 790–797 (2011).
Wilksch, J. J. et al. MrKH, a novel c-di-GMP-dependent transcriptional activator, controls Klebsiella pneumoniae biofilm formation by regulating type 3 fimbriae expression. PLoS Pathog. 7, e1002204 (2011).
Guzman, L.-M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose P BAD promoter. J. Bacteriol. 177, 4121–4130 (1995).
Kalscheuer, R. et al. Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters. Appl. Environ. Microbiol. 72, 1373–1379 (2006).
Harris, L. A. L. S., Skinner, J. R. & Wolins, N. E. Imaging of neutral lipids and neutral lipid associated proteins. Methods Cell Biol. 116, 213–226 (2013).
Ong, N. T. X. & Tabor, J. J. A miniaturized E. coli green light sensor with high dynamic range. Chem. Bio. Chem. 19, 1255–1258 (2018).
Lindner, A. B., Madden, R., Demarez, A., Stewart, E. J. & Taddei, F. Asymmetric segregation of protein aggregates is associated with cellular aging and rejuvenation. Proc. Natl Acad. Sci. USA 105, 3076–3081 (2008).
Winkler, J. et al. Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing. EMBO J. 29, 910–923 (2010).
Paul, K., Nieto, V., Carlquist, W. C., Blair, D. F. & Harshey, R. M. The c-di-GMP binding protein YcgR controls flagellar motor direction and speed to affect chemotaxis by a ‘Backstop Brake’ mechanism. Mol. Cell 38, 128–139 (2010).
Lloyd-Price, J. et al. Asymmetric disposal of individual protein aggregates in Escherichia coli, one aggregate at a time. J. Bacteriol. 194, 1747–1752 (2012).
Rossmann, F. M. et al. The GGDEF domain of the phosphodiesterase PdeB in Shewanella putrefaciens mediates recruitment by the polar landmark protein HubP. J. Bacteriol. 49, https://doi.org/10.1128/JB.00534-18 (2019).
Smith, J. et al. Spatial patterning of P granules by RNA-induced phase separation of the intrinsically-disordered protein MEG-3. eLife 5, 1–18 (2016).
Kulkarni, A. & Extavour, C. G. Convergent evolution of germ granule nucleators: a hypothesis. Stem Cell Res. 24, 188–194 (2017).
Bergé, M. & Viollier, P. H. End-in-Sight: cell polarization by the polygamic organizer PopZ. Trends Microbiol. 26, 363–375 (2018).
Lori, C. et al. Cyclic di-GMP acts as a cell cycle oscillator to drive chromosome replication. Nature 523, 236–239 (2015).
Christen, M. et al. Asymmetrical distribution of the second messenger c-di-GMP on bacterial cell division. Science 328, 1295–1297 (2010).
Zschiedrich, C. P., Keidel, V. & Szurmant, H. Molecular mechanisms of two-component signal transduction. J. Mol. Biol. 428, 3752–3775 (2016).
Tsoi, R. et al. Metabolic division of labor in microbial systems. Proc. Natl Acad. Sci. USA 115, 2526–2531 (2018).
Doong, S. J., Gupta, A. & Prather, K. L. J. Layered dynamic regulation for improving metabolic pathway productivity in Escherichia coli. Proc. Natl Acad. Sci. USA 115, 2964–2969 (2018).
Zhang, H. & Stephanopoulos, G. Co-culture engineering for microbial biosynthesis of 3-amino-benzoic acid in Escherichia coli. Biotechnol. J. 11, 981–987 (2016).
Jin, X. & Riedel-Kruse, I. H. Biofilm Lithography enables high-resolution cell patterning via optogenetic adhesin expression. Proc. Natl Acad. Sci. USA 115, 3698–3703 (2018).
Ojima, Y., Nguyen, M. H., Yajima, R. & Taya, M. Flocculation of Escherichia coli cells in association with enhanced production of outer membrane vesicles. Appl. Environ. Microbiol. 81, 5900–5906 (2015).
Girgis, H. S., Liu, Y., Ryu, W. S. & Tavazoie, S. A comprehensive genetic characterization of bacterial motility. PLoS Genet. 3, 1644–1660 (2007).
Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000).
Acknowledgements
The authors would like to thank J. Tabor for providing plasmids related to the light-inducible transcription activation system. N. Ward provided reagents and equipment. K. Helm and C. Childs, from the Flow Cytometry Shared Resource at the University of Colorado Cancer Center, were supported by the Cancer Center Support Grant No. P30CA046934. This work was supported by School of Energy Resources at the University of Wyoming and by the National Institutes of Health under award numbers 2P20GM103432 and R01GM118792.
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N.V.M. conceived and performed experiments, collected and analyzed data and contributed to the writing of the manuscript. A.F. had a similar role relating to the development of the c-di-GMP biosensor. M.G. guided research strategies and edited the manuscript. G.R.B. conceived experiments, guided research strategies and wrote the manuscript.
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Supplementary Table 1, Supplementary Figures 1–10, Supplementary Note 1
Supplementary Video 1
Asymmetric cell division of YhjH-mChy-PopZ positive cells bearing plasmids cells bearing plasmid pBad-YmP. Same conditions as Fig. 1c. This experiment was performed three times and produced similar results.
Supplementary Videos 2
Examples of asymmetric cell division by YhjH-mChy-PopZ positive cells bearing plasmids pAC-YC-YmP-S and pBad-MrkA-rbs-GFP. Same conditions as Supplementary Video 1 and Fig. 3d. This experiment was performed three times and produced similar results.
Supplementary Videos 3
Examples of asymmetric cell division by YhjH-mChy-PopZ positive cells bearing plasmids pAC-YC-YmP-S and pBad-MrkA-rbs-GFP. Same conditions as Supplementary Video 1 and Fig. 3d. This experiment was performed three times and produced similar results.
Supplementary Video 4
Phase contrast movie of cells exposed to 4 h of pulsed green light, in presence of 0.2% of arabinose. Same conditions as Fig. 4f,g, left panels. This experiment was performed more than three times and produced similar results.
Supplementary Video 5
mChy fluorescence movie of cells exposed to 4 h of pulsed green light, in presence of 0.2% of arabinose. Same conditions as Fig. 4f,g, left panels. This experiment was performed three times and produced similar results.
Supplementary Video 6
Phase contrast movie of cells exposed to 4 h of constant red light, in presence of 0.2% of arabinose. Same conditions as Fig. 4f,g, right panels. This experiment was performed more than three times and produced similar results.
Supplementary Video 7
mChy fluorescence movie of cells exposed to 4 h of constant red light, in presence of 0.2% of arabinose. Same conditions as Figure 4f,g, right panels. This experiment was performed three times and produced similar results.
Supplementary Video 8
Phase contrast movie of wild-type (MG1655) cells. This experiment was performed more than three times and produced similar results.
Supplementary Video 9
Phase contrast movie of ∆motA-motB strain. This experiment was performed more than three times and produced similar results.
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Mushnikov, N.V., Fomicheva, A., Gomelsky, M. et al. Inducible asymmetric cell division and cell differentiation in a bacterium. Nat Chem Biol 15, 925–931 (2019). https://doi.org/10.1038/s41589-019-0340-4
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DOI: https://doi.org/10.1038/s41589-019-0340-4
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